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ALTERNATIVE SPLICING OF TYPE II PROCOLLAGEN GENE IN THE DEDIFFERENTIATION OF RAT EPIPHYSEAL
CHONDROCYTES SERIALLY CULTURED IN MONOLAYER
*Kim, T Kyun; +*Kim, H Joong (A-Korea Educational Ministry); *Park, J Sun; *Lee, M Chul; *Seong, S Cheol
+*Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul, Korea. 28 Yongon-Dong, Chongno-Gu, Seoul, 110-744, Korea, 82-2760-2970, Fax: 82-2-764-2718, [email protected]
Introduction: Release of chondrocytes from their matrix, with the subsequent
growth in monolayer culture, results in dedifferentiation of the chondrocytes
(1). Type II procollagen is synthesized in two forms (types IIA and IIB),
generated by the alternative splicing involving exon 2 of its precursor mRNA
(2). The purpose of this study was to determine whether alternative splicing of
type II procollagen pre-mRNA occurs in dedifferentiation of chondrocytes in
monolayer culture, and whether the morphologic change of chondrocytes
during the dedifferentiaton is linked to this alternative splicing. We serially
cultured rat epiphyseal chondrocytes for eight weeks in monolayer, and
investigated the expression patterns of the two forms of type II procollagen
mRNA according to the duration of the culture period.
Methods: Chondrocytes were released from the epiphyseal cartilage of distal
femurs of ten-day-old Sprague-Dawley rats by collagenase digestion and were
subcultured in monolayer for eight weeks. Total RNA was weekly extracted
from the cultured chondrocytes. Reverse transcription-polymerase chain
reaction (RT-PCR) was done with three sets of primers: primers from exon 1
and exon 5 of the rat type II collagen sequence for relative amount of both
transcripts (IIA and IIB), primers from the triple helical domain for total
amount of type II procollagen mRNA, and primers from both ends of exon 2
for type IIA transcript. To confirm the quantitative evaluation of RT-PCR,
northern analysis of total type II and type IIA mRNA was performed with
cDNA probe constructed in triple helix and cDNA probe from exon 2. For in
situ hybridization of the two forms of type II transcript, chondrocytes were
cultured on the chamber slide. DIG-labeled RNA probe and DIG-labeled
oligonucleotide probe were used to detect type IIA and type IIB mRNA,
respectively. A sense probe was applied as a control for false positive signal.
Results: RT-PCR revealed that type IIA and type IIB procollagen transcripts
were expressed in a distinct way according to the duration of the culture
period (Fig. 1). Both transcripts peaked on the day of cell isolation, but the
expression level of type IIB procollageb mRNA was much higher than that of
type IIA procollagen mRNA. The expression of type IIA form decreased
gradually, whereas the expression of type IIB form decreased so rapidly that
the ratio of type IIB and type IIA transcripts was reversed in the second week
of the culture. Consistent with the results of RT-PCR, northern analysis
showed that the expression of type IIA procollagen, as well as total type II
procollagen peaked on the day of cell isolation (Fig. 2). The expression of
type IIA procollagen decreased gradually, whereas the expression of total type
II procollagen decreased so rapidly that no differences were detectable after
one week. In situ hybridization of type IIA mRNA showed that all cells
expressed type IIA procollagen mRNA throughout the culture period, and that
the cells differed in their expression level according to cell morphology (Fig.
3). All of the freshly isolated chondrocytes revealed positive signals in their
cytoplasm. On the second day, although all of the cells appeared to have
positive signals, the small round cells showed stronger signals than the
medium-sized polygonal cells. On the seventh day, all three sub-populations
revealed positive signals. However, the small round cells showed the strongest
signals, followed by the medium-sized polygonal cells and then the large
fibroblastic cells. In the second week, the situation was the same as on the
seventh day, but the greatest number of cells became large fibroblastic cells
with weak signals. From the third to the eighth week, these large fibroblastic
cells showed weakly positive signals in the cytoplasm. In situ hybridization of
type IIB mRNA showed quite different results from in situ hybridization of
type IIA mRNA. Type IIB procollagen was not expressed in large fibroblastic
cells. Until the second week, it was only expressed in the small, round cells
and in the medium-sized polygonal cells. Like type IIA procollagen, all of the
freshly isolated chondrocytes showed positive signals. However, on the
second day and on the seventh day, the small round cells showed stronger
signals than the medium-sized polygonal cells. In the second week, the
medium-sized polygonal cells and the large fibroblastic cells did not show any
positive signals, while the small round cells showed weak signals. From the
third to the eighth week, no positive signal was detected.
Discussion: These findings indicate that alternative splicing of type II
procollagen pre-mRNA does occur and is regulated in dedifferentiation of
chondrocytes in monolayer culture, and that the morphologic change of
chondrocytes during the dedifferentiaton is linked to this alternative splicing.
Our results support the role of type IIB procollagen as a specific marker for
the chondrocytic phenotype.
Total II
β-actin
Type IIA
β-actin
Type IIA
Type IIB
β-actin
0
1
2
3
4
5
Weeks in culture
6
7
8
Fig. 1. RT-PCR analysis of alternative splicing of type II procollagen gene.
Target
Gene
Exposure
Time
Total II
3 hours
Type IIA
3 days
β-actin
3 hours
0
1
2
3
4
5
Weeks in culture
6
7
8
Fig. 2. Northern analysis of total type II mRNA and type IIA mRNA.
IIA: 0 d
IIA: 1 w
IIA: 2 w
IIB: 0 d
IIB: 1 w
IIB: 2 w
Fig. 3. In situ hybridization of type IIA mRNA and type IIB mRNA.
References: 1) Archer et al. J Cell Sci 97:361-371, 1990
2) Ryan MC, Sandell LJ. J Biol Chem 265:10334-10339, 1990
Acknowledgements: Korea Education Ministry Grant (97-019-D00087)
Poster Session – Collagen - Hall E
47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California
0469